Breast cancer metastasis remains one of the leading causes of death in women. Once tumors have metastasized to distant sites (stage IV) they become incurable. Therefore, there is a critical need for new therapies to treat metastatic tumors. Our long-term goal is to understand the molecular mechanisms of metastasis so that novel targeted therapeutic strategies can be designed. EMT (epithelial to mesenchymal transition) is a key step for a non-invasive cancer cell to progress to an invasive one. Disruption of cell-cell adhesion via loss of E-cadherin (E-cad), the defining event for EMT, is often correlated with gain of the matrix metalloproteinases (MMP) that catalyze proteolytic matrix remodeling important for tumor invasion in invasive breast cancer. However, how EMT is regulated during and contributes to breast cancer metastasis are largely unknown. We propose to define novel roles of Kr[unreadable]ppel-like transcription factor 8 (KLF8) in EMT and metastasis. We previously identified KLF8 as a downstream effector of FAK that plays a crucial role in breast cancer metastasis. Recently, we found that KLF8 is important for transformation and is overexpressed in invasive human breast cancer cells, suggesting a role for KLF8 in breast cancer metastasis that is completely uninvestigated. Preliminary studies suggest that KLF8 is a potent inducer of EMT and invasion by repressing E-cad and activating MMP9, MMP2 and MT1-MMP. Furthermore, KLF8 promoted [unreadable]-catenin (beta-cat)/TCF1 nuclear expression and activity. Our in vivo findings using an inducible KLF8 knockdown and xenograft mouse model suggest a critical role for KLF8 in supporting invasive tumor growth and micrometastasis to the lungs. In addition to these functional data, we analyzed invasive tumor tissue samples from patients and demonstrated that KLF8 levels were well correlated with loss of E-cad and the tumor invasive potential. These results strongly support our hypothesis that KLF8 promotes human breast cancer metastasis by initiating EMT and invasion via repression of E-cad and activation of the MMPs. To test this, we propose three specific aims using a 3D culture model, a mouse model and our MCF-10A cells expressing inducible KLF8 and MDA-MB-231 cells expressing inducible KLF8 shRNA. In Aim 1, we will determine the role of E-cad and the MMPs in KLF8-induced EMT and invasion by genetically modifying these proteins. In Aim 2, we will identify the molecular mechanisms by which E-cad, the MMPs, beta-cat/TCF1 and integrin-FAK signaling interplay to regulate KLF8-induced EMT and invasion using genetic, biochemical or pharmacological approaches. In Aim 3, we will determine the role and mechanisms for KLF8 and its interaction with other EMT-inducing factors in the regulation of invasive tumor growth and metastasis using a mouse model of human breast cancer. Completion of this study will further advance this field by shedding new lights on mechanisms responsible for breast cancer metastasis which may leads to new therapeutic intervention. PUBLIC HEALTH RELEVANCE: Our lab has been leading the study of KLF8 and has made major contributions to advancing this field of research. Our identification of KLF8 as an inducer of EMT and invasion and a regulator of E-cad and MMPs is significant as KLF8 represents a novel family of EMT-inducing transcription factors. Completion of this study will further advance this field by shedding new light on mechanisms responsible for breast cancer metastasis and providing new targets for novel therapeutic designs.